Metagenomic Discovery of 83 New Human Papillomavirus Types in Patients with Immunodeficiency

Abstract

Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus Gammapapillomavirus were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus Betapapillomavirus. Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts.IMPORTANCE Although some members of the viral family Papillomaviridae cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed Gammapapillomavirus (Gamma) virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known Gamma HPV types and suggest that WHIM syndrome patients are uniquely susceptible to Gamma HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual Gamma HPV skin warts observed in WHIM patients.

Keywords: WHIM syndrome; epidermodysplasia verruciformis; gammapapillomaviruses; metagenomic; next-generation sequencing; plerixafor; skin swabs.

FIG 1

Known taxa in skin swabs from healthy subjects, WHIM patients, and patients with other diseases. Donut graphs show the total numbers of reads of known taxa of interest detected in swab samples from individuals from various categories: healthy lab volunteers (TVMBS), lichen sclerosus (LS) patients, Merkel cell carcinoma (M) patients, EV patients, GATA2 (GA2), DOCK8, and WHIM patients (W or WG). For the WHIM patients, each graph shows the first collected sample of affected nongenital or genital (-Gen) skin for each patient.

FIG 2

HPVs and HPyVs in wart biopsy samples/scrapings. (A and B) Warts from immunologically normal subjects (A) or immunodeficient patients (WILD, idiopathic CD4 lymphopenia [ICL], and WHIM [W] patients) (B) were assessed for the presence of distinct HPV types. Genera are depicted in different colors, with slices of the same color representing different types in the same genus. Blue numbers inside the donut represent the total number of viral types for the sample. In some cases, such as W03, some of the types represented a very small fraction of the whole, and wedges are not discernible. Black numbers on the donut correspond to the HPV type or isolate designation (letters are used to denote not-yet-assigned new HPV types), or human polyomavirus (HPyV) species number. For example, Wart I contained a single type, HPV1 from the Mupapillomavirus genus (Mu). For clarity, only the larger predominant graph segments are annotated with HPV type designations. Some patients had biopsy specimens of multiple warts, and each is shown individually.

FIG 3

Phylogenetic analysis of reported HPV types. (A and B) Protein sequences from L1 (A) or E1 (B) were used to construct phylogenetic trees. At least one HPV type from each previously known species (black font) was analyzed along with each of the 83 complete HPV genomes catalogued in the current study. Asterisks were used to show known representative species that did not fit due to the figure’s space restrictions (counterclockwise, Beta 3-HPV49, Gamma 20-HPV163, Gamma 21-HPV167, and Gamma 2-HPV48). HPV genera are depicted by different colors as follows: orange, Alpha; red, Beta; blue, Gamma; pink, Mu; green, Nu. Dotted lines are not significant; they were used to indicate HPV type names. Arrows indicate potential new species.

FIG 4

Comparison of viral diversity among skin swabs of patients. (A to D) Analysis of pooled swab samples from affected (warts, rashes, etc.) and healthy skin from the earliest single time point for MCC patients (A), EV patients (B), a GATA2, a DOCK8 and an ICL patient (originally classified as EV-like) (C), and WHIM patients (D). Blue numbers inside the donut represent the numbers of viral types. Black numbers on the donut represent predominant HPV types (letters are used to denote not-yet-assigned new HPV types).

FIG 5

HPV typing for three individuals on plerixafor therapy. Analysis of samples from WHIM patients during long treatment. Each donut represents pooled samples from several anatomical locations. Samples from patient W06 are from the same time point, the first donut contains swabs from the following sites: right (R) 4th finger, R wrist, and an oral lesion; the second donut represents pooled swabs from the R 2nd finger, R forearm, and R palm. Samples from patient W20 are from different time points: (i) R calf, R ankle, left (L) top of foot, L thigh, and R sole of foot; (ii) normal skin of the abdomen; (iii) L palm and right foot, (iv) L eye cantus; (v) R leg follicular lesions; (vi) R leg open wound. Samples from patient W27 are from the following sites: (i) R toes, R dorsum of foot, R middle finger, R ankle, R jaw, L hand; (ii) R middle finger, L ring finger, L hand, R toes, R calf; (iii) R sole; (iv) L hand periungal. The first available time points for W20 and W27 were first shown in Fig. 4, and are also added here for comparison. Blue numbers inside the donut represent the numbers of viral types. Black numbers on the donut represent predominant HPV types (letters are used to denote not-yet-assigned new HPV types).